Evaluation of the Effectiveness of Crotoxin as an Antiseptic against Candida spp. Biofilms.

The growing number of oral infections caused by the Candida species are becoming harder to treat as the commonly used antibiotics become less effective. This drawback has led to the search for alternative strategies of treatment, which include the use of antifungal molecules derived from natural products. Herein, crotoxin (CTX), the main toxin of Crotalus durissus terrificus venom, was challenged against Candida tropicalis (CBS94) and Candida dubliniensis (CBS7987) strains by in vitro antimicrobial susceptibility tests. Minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and inhibition of biofilm formation were evaluated after CTX treatment. In addition, CTX-induced cytotoxicity in HaCaT cells was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay. Native CTX showed a higher antimicrobial activity (MIC = 47 µg/mL) when compared to CTX-containing mouthwash (MIC = 750 µg/mL) and nystatin (MIC = 375 µg/mL). Candida spp biofilm formation was more sensitive to both CTX and CTX-containing mouthwash (IC100 = 12 µg/mL) when compared to nystatin (IC100 > 47 µg/mL). Moreover, significant membrane permeabilization at concentrations of 1.5 and 47 µg/mL was observed. Native CTX was less cytotoxic to HaCaT cells than CTX-containing mouthwash or nystatin between 24 and 48 h. These preliminary findings highlight the potential use of CTX in the treatment of oral candidiasis caused by resistant strains.

Intradermal Application of Crotamine Induces Inflammatory and Immunological Changes In Vivo.

Crotamine is a single-chain polypeptide with cell-penetrating properties, which is considered a promising molecule for clinical use. Nevertheless, its biosafety data are still scarce. Herein, we assessed the in vivo proinflammatory properties of crotamine, including its local effect and systemic serum parameters. Sixty male Wistar rats were intradermically injected with 200, 400 and 800 µg crotamine and analyzed after 1, 3 and 7 days. Local effect of crotamine was assessed by determination of MPO and NAG activities, NO levels and angiogenesis. Systemic inflammatory response was assessed by determination of IL-10, TNF-α, CRP, NO, TBARS and SH groups. Crotamine induced macrophages and neutrophils chemotaxis as evidenced by the upregulation of both NAG (0.5⁻0.6 OD/mg) and MPO (0.1⁻0.2 OD/mg) activities, on the first and third day of analysis, respectively. High levels of NO were observed for all concentrations and time-points. Moreover, 800 µg crotamine resulted in serum NO (64.7 µM) and local tissue NO (58.5 µM) levels higher or equivalent to those recorded for their respective histamine controls (55.7 µM and 59.0 µM). Crotamine also induced a significant angiogenic response compared to histamine. Systemically, crotamine induced a progressive increase in serum CRP levels up to the third day of analysis (22.4⁻45.8 mg/mL), which was significantly greater than control values. Crotamine (400 µg) also caused an increase in serum TNF-α, in the first day of analysis (1095.4 pg/mL), however a significant increase in IL-10 (122.2 pg/mL) was also recorded for the same time-point, suggesting the induction of an anti-inflammatory effect. Finally, crotamine changed the systemic redox state by inducing gradual increase in serum levels of TBARS (1.0⁻1.8 µM/mL) and decrease in SH levels (124.7⁻19.5 µM/mL) throughout the experimental period of analysis. In summary, rats intradermally injected with crotamine presented local and systemic acute inflammatory responses similarly to histamine, which limits crotamine therapeutic use on its original form.

Crotoxin from Crotalus durissus terrificus venom: In vitro cytotoxic activity of a heterodimeric phospholipase A2 on human cancer-derived cell lines.

Crotoxin (CTX), a heterodimeric phospholipase present in venom of snakes of the genus Crotalus, has demonstrated a broad spectrum of pharmacological properties, such as antimicrobial, hemostatic, and antitumoral. However, the precise mechanism of its cytotoxicity and antitumoral properties remains to be determined. Therefore, in the present study, we isolated crotoxin (F1 CTX) through two steps DEAE-Sepharose and Heparin-Sepharose FF chromatography. The C-terminal sequence of the A- and B-chain protein fragment was determined by LC-MS/MS mass spectrometry, which showed 100% identity to crotoxin structure. In order to investigate its cytotoxic effects, we demonstrated that the F1 CTX fraction at 0-30 µg/mL concentrations for 72 h presented a heterogeneous response profile on nine human cancer-derived cell lines from four tumor types (pancreatic, esophagus, cervical cancer, and glioma). The glioma (GAMG and HCB151) and pancreatic (PSN-1 and PANC-1) cancer cells showed a higher sensitivity with IC50 of <0.5, 4.1, 0.7 and < 0.5 µg/mL, respectively. Conversely, F1 CTX does not reduce the viability of normal cells. On the other hand, cervical (SiHa) and esophagus (KYSE270) cancer cell lines presented higher resistance, with IC50 higher than 30.2 and 8.7 µg/mL, respectively. Moreover, F1 CTX did not affect cell cycle distribution under the conditions evaluated and seems to be more cytotoxic than cytostatic. The pro-apoptotic effect of F1 CTX treatment was demonstrated in glioma (HCB151) cell line. In addition, crotoxin revealed a potential to initiate cell responses such as DNA damage in glioma (HCB151) and pancreatic cancer by H2AX activity induction. Conversely, F1 CTX does not reduce the viability of normal cells. Importantly, the comparison of F1 CTX effect with standard chemotherapeutic agents demonstrated a greater cytotoxic potential in the majority of tumor types (glioma, pancreatic, and cervical cancer). On the other hand, F1 CTX was less cytotoxic in esophageal cell lines compared to the gemcitabine agent used in clinical practice. Therefore, this work showed that F1 CTX has a cytotoxic activity and pro-apoptotic potential, contributing to the knowledge about the F1 crotoxin properties as well as its possible use in cancer research, particularly in glioma and pancreatic cancer cell lines.

Mechanistic insights into functional characteristics of native crotamine.

The chemical composition of snake venoms is a complex mixture of proteins and peptides that can be pharmacologically active. Crotamine, a cell-penetrating peptide, has been described to have antimicrobial properties and it exerts its effects by interacting selectively with different structures, inducing changes in the ion flow pattern and cellular responses. However, its real therapeutic potential is not yet fully known. Bearing in mind that crotamine is a promising molecule in therapeutics, this study investigated the action of purified molecule in three aspects: I) antibacterial action on different species of clinical interest, II) the effect of two different concentrations of the molecule on platelet aggregation, and III) its effects on isolated mitochondria. Crotamine was purified to homogeneity in a single step procedure using Heparin Sepharose. The molecular mass of the purified enzyme was 4881.4 Da, as determined by mass spectrometry. To assess antibacterial action, changes in the parameters of bacterial oxidative stress were determined. The peptide showed antibacterial activity on Escherichia coli (MIC: 2.0 µg/µL), Staphylococcus aureus (MIC: 8-16 µg/µL) and methicillin-resistant Staphylococcus aureus (MIC: 4.0-8.0 µg/µL), inducing bacterial death by lipid peroxidation and oxidation of target proteins, determined by thiobarbituric acid reactive substances and sulfhydryl groups, respectively. Crotamine induced increased platelet aggregation (IPA) at the two concentrations analyzed (0.1 and 1.4 µg/µL) compared to ADP-induced aggregation of PRP. Mitochondrial respiratory parameters and organelle structure assays were used to elucidate the action of the compound in this organelle. The exposure of mitochondria to crotamine caused a decrease in oxidative phosphorylation and changes in mitochondrial permeability, without causing damage in the mitochondrial redox state. Together, these results support the hypothesis that, besides the antimicrobial potential, crotamine acts on different molecular targets, inducing platelet aggregation and mitochondrial dysfunction.

Isolation, characterization and screening of the in vitro cytotoxic activity of a novel L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom on human cancer cell lines.

An L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom was purified to homogeneity in a two-step procedure using molecular exclusion on Sephadex G-75, followed by Phenyl Sepharose FF chromatography. The molecular mass of the purified enzyme was 113 kDa, as determined by SDS-PAGE under reducing conditions. LAAOcdt showed amino acid homology to other L-amino acid oxidases isolated from different snake venoms. The comparative analysis of the internal peptide sequences of the NNPGILEYPVKPSEEGK fragments by LC-MS/MS spectrometry revealed 100% identity with C. durissus cumanensis LAAO. The purified protein catalyzed the oxidative deamination of L-amino acids, and the most specific substrates were L-Tyr and L-Phe. The enzyme presented optimum activity at pH 7.4 and at 44 °C. LAAOcdt also showed hemolytic activity (0.6-20 µg/µL) and induced both the formation plasma clots (5-100 µg/µL) and platelet aggregation (2.5 × 10(-3), 5.0 × 10(-3) and 10 × 10(-3) µg/mL), as well as bactericidal activity (2.5-10 µg/µL) against Staphylococcus aureus. Moreover, LAAOcdt exhibited cytotoxicity in distinct cancer cell lines, which presented a heterogeneous response profile. The mean IC50 value was 10.5 µg/mL. Glioma and pancreatic carcinoma cells were the most sensitive cell lines; they showed mean IC50 values of 7.2 µg/mL and 7.4 µg/mL, respectively. The exposure of the drug-sensitive cells to LAAOcdt for 24 h upregulated activated p-H2AX and efficiently decreased P42/P44 (ERK) activation in glioma cells (HCB151), which suggested an anti-proliferative effect. In addition, increased p21 expression was observed in SiHa cells, which showed a resistant phenotype. On the other hand, the flow cytometry and immunoblotting analyses showed that the enzyme did not induce cancer cell apoptosis. These results suggest that another cell death mechanism might contribute to the LAAOcdt-induced cytotoxicity. Taken together, this work may help to elucidate the function and structure of LAAOcdt by providing the basis for further investigations on its efficacy in cancer treatment.

Anti-hemorrhagic effect of hydro-alcoholic extract of the leaves of Mikania glomerata in lesions induced by Bothrops jararaca venom in rats.

PURPOSE: To investigate the effects of hydroalcoholic leaf extract of Mikania glomerata Spreng (Asteraceae) on the activity of Bothrops jararaca snake venom in Wistar rats. METHODS: Fifty four rats Wistar were divided into six groups of nine animals in each: control treated with saline; control treated with B. jararaca venom; control treated with M. glomerata extract; B. jararaca venom incubated with M. glomerata extract at proportions of 1:1, 1:2, and 1:4. RESULTS: Histopathological and morphometric analysis showed that intradermal administration of snake venom incubated with the hydroalcoholic extract at proportions of 1:1, 1:2 and 1:4 promoted a significant reduction in the number of inflammatory cells and a marked decrease in edema after the third hour. There was also a significant reduction in the intensity of the hemorrhagic halo in animals receiving the snake venom incubated with the extract, with the observation of a progressive and parallel inhibition with increasing proportion of M. glomerata. CONCLUSION: The Mikania glomerata hydroalcoholic extract exerted effective anti-inflammatory and antihemorrhagic activity against the effects induced by Bothrops jararaca snake venom.

Anti-hemorrhagic effect of hydro-alcoholic extract of the leaves of Mikania glomerata in lesions induced by Bothrops jararaca venom in rats

To investigate the effects of hydroalcoholic leaf extract of Mikania glomerata Spreng (Asteraceae) on the activity of Bothrops jararaca snake venom in Wistar rats. METHODS: Fifty four rats Wistar were divided into six groups of nine animals in each: control treated with saline; control treated with B. jararaca venom; control treated with M. glomerata extract; B. jararaca venom incubated with M. glomerata extract at proportions of 1:1, 1:2, and 1:4. RESULTS: Histopathological and morphometric analysis showed that intradermal administration of snake venom incubated with the hydroalcoholic extract at proportions of 1:1, 1:2 and 1:4 promoted a significant reduction in the number of inflammatory cells and a marked decrease in edema after the third hour. There was also a significant reduction in the intensity of the hemorrhagic halo in animals receiving the snake venom incubated with the extract, with the observation of a progressive and parallel inhibition with increasing proportion of M. glomerata. CONCLUSION: The Mikania glomerata hydroalcoholic extract exerted effective anti-inflammatory and antihemorrhagic activity against the effects induced by Bothrops jararaca snake venom.